Monoclonal antibodies were once thought to be an ideal way to target malignant tissues, by delivering a killing agent, while leaving healthy tissue intact. However, their clinical potential is limited due to the need to covalently couple the killing agent to the monoclonal antibody. Thus, in an effort to alleviate such limitations, bispecific antibodies were developed, which remain bivalent, but are specific for a target cell on one arm of the antibody and a killing agent on the other arm. The killing agent can be a toxin, a drug, a chelated radioisotope, or, more likely, a cytotoxic effector cell.
Monoclonal antibodies can also show therapeutic activity against specific cells, e.g., malignant tissues based on the interaction of the Fc portion of the antibody heavy chain with other components of the immune system, such as the complement cascade or by binding to Fcγ receptors or various cytotoxic effector cell types.
Another means of effecting cell death comprises inducing the cross-linking of membrane antigens. Previous studies have indicated that antibody cross-linking of membrane B-cell markers (e.g., surface IgM, Valentine et al., Eur. J. Immunol. 22:3141 (1992); and MHC class II, Newell et al., PNAS 90:10459 (1993)) can inhibit malignant B cell proliferation and in many cases induce apoptosis (e.g., programmed cell death) in vitro.
Shan et al. (Blood 91:1644-1653) demonstrated that hyper-cross-linking of the CD20 antigen, by using the murine 1 F5 antibody cross-linked with a goat anti-mouse IgG, inhibited growth of several human B-lymphoma cell lines in vitro. Similar results have now been published for both CD19 and CD22 when cross-linking of membrane bound MAb was amplified with a anti-mouse IgG (Chaouchi et al., J. Immunol. 154:3096 (1995)).
It may be possible that hyper cross-linking of these surface membrane markers could augment the existing anti-tumor activities of MAb's like C2B8, a chimeric monoclonal antibody specific for CD20, and increase therapeutic effectiveness. Therefore, molecules that can induce cell death in a pharmaceutically acceptable format would potentially provide an attractive therapeutic agent for immunotherapy of neoplastic disease.
Apparently with that goal in mind, Wolff et al. (Cancer Res. 53:2560-2565 (1993)) and Ghetie (PNAS 94:7509-7514 (1997)) have reported the chemical synthesis of several IgG/IgG homodimers to carcinoma associated surface antigen (BR96 and HER-2). The Ghetie dimers also included antibodies to several human B-cell markers (CD20, CD19, CD21, CD22). In this approach, one portion of the molecule was functionalized using a linker designed to introduce a reactive thiol on the antibody, while the other Ab portion used a linker to introduce a maleimido group. When purified from unreacted linkers and mixed together, the two antibodies complex by formation of a thioether (non-reducible) bridge that links the two IgG molecules, and forming a 300 kDa, tetravalent antibody (C2H2)2g molecule.
However, unfortunately, the yields of the 300 kDa IgG-homodimer were very low (20-25%) and were similar or lower than “spontaneously” formed CD19 homodimer, which ranged from 20-30% (Ghetie et al., PNAS94:7509-7514 (1997)).
Reducing SDS-PAGE gels of purified homodimer showed only a small percentage was linked via a thioether bond, indicating most of the dimers formed using this methodology may have been naturally occurring or mediated through disulfide bridging. Nevertheless, all of the purified dimers were growth inhibitory, although only the anti-carcinoma (Her-2) dimer and not homodimers directed against B cell markers CD19, CD20, CD21, CD22 were reported to be apoptotic. Additionally, the anti-CD19 homodimer was tested in animal models and shown to have anti-tumor activity. However, there is a need in the art for a more efficient method for producing homodimers, in particular for homodimers or heterodimers that are capable of initiating apoptosis, e.g., in proliferating malignant B-cells populations.
In the present invention, two monoclonal antibodies were used: a mouse/human chimeric antibody specific for CD20 (C2B8), and a Primatized® antibody specific for CD23 (p5E8). Low grade and aggressive B-cell lymphomas express the B cell antigens CD20 and CD23. CD20 is a non-glycosylated 35 kDa B-cell membrane protein associated with intracellular signaling, B-cell differentiation and calcium channel mobilization (Clark et al., Adv. Cancer Res. 52:81-149 (1989); Tedder et al., Immunol. Today 15:450-54 (1994)). The antigen appears as an early marker of the human B-cell lineage, and is ubiquitously expressed at various antigen densities on both normal and malignant B cells. However, the antigen is absent on stem cells or pre B cell populations, as well as on the fully matured plasma cell, making it a good target for antibody mediated therapy. CD23 is the low affinity receptor for IgE. Antibodies to CD23 have been suggested to be useful for treating allergic and inflammatory responses. In fact, IDEC Pharmaceuticals, Inc., the assignee of this application, has an application pending relating to the use of an anti-CD23 antibody of the IgG1 isotype for therapeutic usage. Of importance herein, CD23 is expressed on B-cells, and particularly by B-cell lymphoma cells.
While only a small fraction of the CD20 antigen is expressed on the surface membrane, MAb's binding to the extracellular domain have had variable activities in promoting or inhibiting B cell function. For example, the anti-CD20 MAb, IF5, was originally shown to activate resting (G0) B-cells into (G1/S/G2) proliferating populations (Clark et al., PNAS, USA, 82:1766-70 (1985)). Additionally, Holder et al. (Eur. J. Immunol. 25:3160-64 (1995) demonstrated that Mab IF5 cross-linking of the CD20 surface antigen protected proliferating tonsular B cells from undergoing apoptosis (programmed cell death) in vitro. In contrast, the anti-CD20 antibody B1 that binds to a different epitope than IFS (Tedder et al., Immunol. Today 15:450 (1994), was not stimulatory for resting B cell populations (Tedder et al., Eur. J. Immunol. 16:881 (1986)).
Despite differences in activity using normal B cell populations, murine anti-CD20 MAb's (e.g., IF5, B1, B20 and 2H7) had no effect on growth inhibition of proliferating human (CD20+) lymphoma cell lines in vitro, but in vivo showed tumor growth inhibition using human lymphoma mouse xenograft models (Press et al, Blood 69:584-591 (1987); Shan et al., Blood 91:1644-1653 (1998); Funakoshi et al., J. Immunol. 19:93-101 (1996); Hooijberg et al., Cancer Res. 55:840-846 (1995); and Ghetie et al., PNAS 94:7509-7514 (1997)). The mechanism mediating anti-tumor activity remains unclear but may be mediated through complement dependent cell killing (CDC) or antibody dependent cell killing (ADCC), both of which are dependent on activation of host cell mechanisms through the Fc portion of the MAb after CD20 binding. Indeed, Funakoshi et al. (J. Immunol. 19:93-101 (1996)) has shown that the anti-tumor activity of 2H7 in vivo was blocked when Fc receptor was blocked or with a F(ab)2 antibody.
The chimeric MAb used in the present invention (C2B8) was developed at IDEC Pharmaceuticals Corporation for treatment of human B cell lymphoma (Reff et al., Blood 83:4350445 (1994)). C2B8 originated from the murine antibody 2B8 and was cloned and expressed as a 150 kDa IgG monomer in Chinese Hamster Ovary cells. MAb C2B8 maintains the 2B8 murine variable region coupled to the human gamma 1 heavy chain and human K light chain constant regions. Like its murine counterparts, C2B8 was not growth inhibitory and does not induce apoptosis of human lymphoma cell lines in vitro, but does demonstrate anti-tumor activity when tested in vivo using murine xenograft animal models.
Chimeric C2B8 efficiently binds human complement, has strong FcR binding, and can efficiently kill human lymphocytes in vitro via both complement dependent (CDC) and antibody dependent (ADCC) mechanisms (Reff et al., Blood 83:435-445 (1994)). C2B8 was also strongly depleting of B cells in human Phase I/II clinical trials, but was nevertheless shown to be safe and effective with most side effects infusion related (Maloney et al., Blood 84:2457-2466 (1994) and Maloney et al., JCO 15(10):3266 (1997)).
The antibody showed an overall response rate of 48% in patients with low grade or follicular lymphoma (McLaughlin et al., JCO, in press). However, the response rate decreased dramatically (34%) in chemo-resistant patients who failed to respond to their last chemotherapy regime (McLaughlin et al., Proc. Am. Soc. Clin. Oncol. 16:16a (Abstr. 55) (1997)). Additionally, the antibody showed poor activity in patients with type A histology or with chronic lymphocytic leukemia (CLL). Therefore, the need to increase the effectiveness of antibody immunotherapy and, specifically, using C2B8 or CD23 antibody therapy remains a high priority in the treatment of human leukemia and lymphoma patients. The anti-CD23 antibody exemplified in the methods herein was also developed by IDEC and is a primatized anti-CD23 antibody of the IgG1 isotype.